Cardiometabolic Syndrome: an Update on Available Mouse Models

Published online: 2020-12-06

Review Article 703

Cardiometabolic Syndrome: An Update on Available Mouse Models

Dimitra Aravani1 Eva Kassi2 Antonios Chatzigeorgiou1,3, Styliani Vakrou1,4,

1 Department of Physiology, Medical School, National and Address for correspondence Antonios Chatzigeorgiou, MD, PhD, Kapodistrian University of Athens, Athens, Greece Department of Physiology, Medical School, National and Kapodistrian 2 Department of Biological Chemistry, Medical School, National and University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece Kapodistrian University of Athens, Athens, Greece (e-mail: [email protected]). 3 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital and Faculty of Medicine Carl Gustav Carus of TU Dresden, Styliani Vakrou, MD, PhD, Department of Physiology, Medical School, Dresden, Germany National and Kapodistrian University of Athens, 75 Mikras Asias 4 Department of Cardiology, “Laiko” General Hospital, Athens, Greece Street, 11527 Athens, Greece (e-mail: [email protected]).

Thromb Haemost 2021;121:703–715.

Abstract Cardiometabolic syndrome (CMS), a disease entity characterized by abdominal obesity, insulin resistance (IR), hypertension, and hyperlipidemia, is a global epidemic with approximately 25% prevalence in adults globally. CMS is associated with increased risk for cardiovascular disease (CVD) and development of diabetes. Due to its multifactorial etiology, the development of several animal models to simulate CMS has contributed significantly to the elucidation of the disease pathophysiology and the design of therapies. In this review we aimed to present the most common mouse models used in the research of CMS. We found that CMS can be induced either by genetic manipulation, leading to dyslipidemia, lipodystrophy, obesity and IR, or obesity and hypertension, or by administration of specific diets and drugs. In the last decade, the ob/ob and / / db/db mice were the most common obesity and IR models, whereas Ldlr and Apoe were widely used to induce hyperlipidemia. These mice have been used either as a single transgenic or combined with a different background with or without diet Keywords treatment. High-fat diet with modifications is the preferred protocol, generally leading ► cardiometabolic to increased body weight, hyperlipidemia, and IR. A plethora of genetically engineered syndrome mouse models, diets, drugs, or synthetic compounds that are available have advanced ► obesity the understanding of CMS. However, each researcher should carefully select the most ► insulin resistance appropriate model and validate its consistency. It is important to consider the ► hyperlipidemia differences between strains of the same animal species, different animals, and most ► mouse model importantly differences to human when translating results. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. Introduction ation between abdominal adiposity and increased risk of type 2 diabetes and CVD.2 CMS is now defined as a disease entity The cardiometabolic syndrome (CMS) was first described over characterized by the simultaneous occurrence of at least three 90 years ago by Kylin as a combination of hyperglycemia, of the following medical conditions: abdominal obesity, raised hypertension, and gout.1 Later on, Vague pointed out an associ- plasma glucose, elevated blood pressure, raised triglycerides (TGs) or reduced high-density lipoprotein (HDL) cholesterol.3 These authors contributed equally to this work as senior authors. Additional components of CMS which are interconnected

received © 2020. Thieme. All rights reserved. DOI https://doi.org/ June 3, 2020 Georg Thieme Verlag KG, 10.1055/s-0040-1721388. accepted after revision Rüdigerstraße 14, ISSN 0340-6245. October 24, 2020 70469 Stuttgart, Germany published online December 6, 2020 704 Cardiometabolic Syndrome: An Update Aravani et al.

through cytokine release from adipose tissue are (1) a proin- criteria: original research articles and those reporting the flammatory state, suggested by elevated C-reactive protein main aspects of CMS. Similar number (277) was obtained (CRP) and (2) a prothrombotic state, characterized by increased using Scopus as a search engine. To complement our search, plasma plasminogen activator inhibitor-1 and fibrinogen.4 we also investigated the MGI database; we found 169 mouse Microalbuminuria, increased inflammation/oxidative stress, models linked to CMS syndrome and the most relevant endothelial dysfunction, nonalcoholic steatohepatitis, abnor- models are included in this review. malities in the coagulation system, and enhanced cardiovascular tissue renin–angiotensin–aldosterone system (RAAS) 5,6 Overview of the Best Characterized Mouse contribute to the pathophysiology of CMS. The accumulation Models of the above metabolic and underlying risk factors promotes the development of atherosclerotic CVD and increases the risk for There are three main ways of modeling CMS in mice. These – type 2 diabetes, stroke, and cognitive impairment.6 8 are genetic manipulation, diet/drug-induced protocols, and The prevalence of metabolic syndrome in United States the combination of any of the above. has been estimated to be around 35% in both sexes of adults, whereas it increases to around 50% in the age group over Genetic CMS Mouse Models – 60 years old and particularly in women.9 11 Increasingly high The strategies of genetic engineering are focused on alteration prevalence of CMS is seen in populations of different coun- of lipid metabolism, weight regulation, glucose homeostasis, tries, confirming the global burden of the disease.9,12,13 blood pressure regulation, and the suitable combination of the Several prospective cohorts and meta-analysis studies dem- above phenotypes. onstrate that CMS is associated with increased relative risk for cardiovascular outcomes (twofold), diabetes (3.5–5-fold), Mouse Models of Dyslipidemia and all-cause mortality (1.5-fold).7,14 In a longitudinal co- Low-density lipoprotein (LDL) receptor (Ldlr) and apolipopro- hort study of 1 million Chinese, the prevalence of cardiome- tein e (Apoe)-deficient mice are the two best characterized tabolic multimorbidity increased from 2.41 to 5.94% in dyslipidemic models. Their hyperlipidemic profile is due to the 5 years, indicating the rapid progression of cardiometabolic absence of either Ldlr or Apoe, both of which participate in disease.15 cholesterol clearance.22,23 Ldlr/ mice develop moderate Because of the multifactorial nature of this syndrome and hypercholesterolemia (total cholesterol 250 mg/dL) on a the lack of specific treatment,16 it is essential to elucidate the normal diet with lipoprotein profiles similar to those of exact pathophysiologic mechanisms. humans (i.e., elevated LDL) but they are very responsive to Animal models have traditionally been used in research atherogenic diet (1.25% cholesterol diet), developing large for CVDs in an effort to translate mechanistic and therapeutic atherosclerotic lesions and hyperlipidemia. Furthermore, – / findings into human disease.17 19 Yet it is critical and rather when Ldlr mice are placed on a diet with greater than challenging to choose the appropriate model that would best 20% fat content, they also become obese, display insulin reflect the human pathophysiology, since animal models resistance (IR), and impaired glucose tolerance.24 Apoe/ present only a fraction of the CMS features at a given time. mice develop a more severe hyperlipidemia, with an increase Rodent models and particularly mice have been the most in plasma cholesterol levels and TG levels, which leads to popular animal models in the investigation of CMS. This is spontaneous atherosclerosis on a normal diet.23 In many cases, because of their obvious advantages over other animals, which Apoe/ mice do not become obese, nor do they develop IR, include easy handling and relatively easy genetic engineering, even on a high-fat diet (HFD).25,26 However, there has been a short gestation and lifespanperiods, and most importantly high case where Apoe/ mice fed HFD (60% fat) for 17 weeks genetic and physiological similarity to humans.20 Moreover, displayed increased body weight, glucose intolerance, and they are responsive to experimental diets as they develop an increase in systemic inflammation, which indicates that central obesity, hyperglycemia, and hypertension, some of modulation of the feeding protocol can have a significant 27

the main characteristics of the CMS. The importance and biological effect. In general, as summarized by the Jackson This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. popularity of mouse models are also reflected by the numerous Laboratory and the Mouse Phenome Database which have studies that used mice as the reference animal. The database phenotyped 8-week old male and female Apoe and Ldlr null mouse genome informatics (MGI) has catalogued 169 mouse mice against C57BL/6J mice after 6, 10, and 14 weeks of normal models matching the phenotype “metabolic syndrome,” which diet (6% fat), these transgenics develop a range of cardiovascu- are linked to 265 references. Moreover, genetic variation iden- lar phenotypes. These include elevated plasma cholesterol and tified in mice has been shown to affect the corresponding TG for both knockouts as well as increased LDL, TG, and ApoB human phenotypes.21 levels in male Ldlr/. Caution should be taken however when The aim of this review is to summarize the best charac- comparing absolute lipid concentrations between Apoe/ and terized mouse models for CMS research and present newer Ldlr/ as various factors could lead to misinterpretations, models that have been described during the past 10 years. such as the mechanism of function for each model and differ- For this purpose, we conducted a PubMed search using the ences in the major plasma lipoproteins. Specifically, in Apoe/ terms cardiometabolic AND mouse models, with a time filter mice the accumulating particles are predominantly apoB48- of 10 years. This search returned 292 results, of which we containing cholesterol ester particles, whereas in Ldlr/ the selected the most appropriate ones using the following major lipoprotein is the ApoB100-containing LDL. Other

Thrombosis and Haemostasis Vol. 121 No. 6/2021 © 2020. Thieme. All rights reserved. Cardiometabolic Syndrome: An Update Aravani et al. 705 factors affecting the absolute lipid concentrations include sex, Obesity and Hypertension Models genetic variations, and differences in gut microbiota.28 Adiponectin is another protein that has been associated with obesity and obesity-related diseases in humans.44 It is secreted Mouse Models of Obesity and IR by adipocytes and low levels are detected in obesity, which can Visceral obesity is one of the requirements for CMS and has be reversed during weight loss. Adipo/ mice fed high fat-high been positively associated with IR and hyperglycemia.29 In sucrose diet for short periods (2–4 weeks) develop elevated agreement with human studies, most of the obesity mouse glucose, insulin and free fatty acids, plasma TNF-a, and hyper- models manifest IR. Leptin deficiency is one of the gold tension.45,46 An established model of systemic lupus eryth- standard models. A spontaneous mutation in leptin gene ematosus (NZBWF1) was described by Ryan et al in 2006 as led to the description of ob/ob mice.30 Leptin deficiency potentially important model to study obesity and IR. The results in increased food intake, extreme obesity, and NZBWF1 mouse phenotype showed increased weight, plasma reduced energy expenditure. The effect on glucose metabo- insulin, but not glucose levels, increased plasma leptin, renal lism is strain-dependent. A C57BL/6J background is charac- adipose tissue macrophage infiltration, and hypertension.47 terized by mild hyperglycemia compared with severely diabetic ob/ob mice on the C57BL/KsJ background. Moreover Nonobese-Lipodystrophic Models ob/ob mice show increased HDL and reduced LDL, hence they The lipodystrophic A-ZIP F-1 and the aP2-SREBP-1c mice are are protected from atherosclerosis and they are also resistant nonobese mouse models, with restricted adipose capacity to hypertension.31,32 A single autosomal mutation in the concerning mainly the white adipose tissue (WAT) and may leptin receptor33 causes almost identical phenotypes be used in the study of nonobese CMS and genetic forms of to leptin deficiency in those mice (db/db) which develop lipodystrophies.48,49 The A-ZIP F-1 mice were generated by obesity, hyperinsulinemia, hyperglycemia, and increased enhancing the expression of A-ZIP/F (dominant negative total cholesterol (predominantly LDL/HDL particles) in the protein of C/EBP transcription factors that regulate growth absence of hypertension.34 An additional model with altered and differentiation of adipocytes) by using an adipose-spe- leptin signaling is the POUND mouse generated by Charles cific P2 enhancer promote. This model, which has complete River, which lacks all leptin receptor isoforms. When fed deficiency of WAT, shows that fat ablation can cause liver Purina diet they became hyperinsulinemic and hyperglyce- steatosis and diabetes, with reduced leptin and elevated mic, which was accompanied by increased leptin and cho- serum glucose, insulin, free fatty acids and TG, and elevated lesterol levels.35 Another model used in CMS research is the blood pressure.48,50 The transgenic aP2-SREBP-1c mice, agouti yellow obese mouse (Ay/a). In the brain the secreted which overexpress sterol regulatory element binding pro- agouti protein antagonizes the binding of the anorexigenic α- teins (SREBP) in adipose tissue, have decreased WAT but have melanocyte-stimulating hormone (α-MSH) to its receptor hypertrophic brown adipose tissue containing cells resem- (melanocortin 4 receptor, MC4-R). α-MSH reduces food bling immature WAT. These mice are characterized by intake, increases energy expenditure, and regulates glucose hyperglycemia, hepatic steatosis, and hyperinsulinemia po- metabolism, thus spontaneous mutations that occur in tentiated by secreted frizzled-related protein.49,51 the agouti gene lead to disruption of these regulatory To better recapitulate the various aspects of the metabolic mechanisms. Agouti mice present adult-onset obesity, syndrome combinations such as obesity and hyperlipidemia, hyperinsulinemia, glucose intolerance, hyperglycemia, and obesity and hypertension have also been employed and hypertension, which establishes it as a useful model of reviewed in detail by Kennedy et al.34 ►Table 1 summarizes CMS.36,37 As mentioned above, hypothalamic MC4-R-medi- the mouse models described above. ated signaling plays a role in food intake and energy homeostasis; therefore, targeted disruption of MC4-R was used as Diet-Induced CMS Mouse Models an alternative approach. The human relevance of this model Numerous dietary interventional studies have been conducted is supported by studies which revealed that MC4-R muta- to study the pathophysiology of CMS. As with the genetic 38,39 MC4-R tions are associated with hereditary obesity. The models these include either a single experimental diet or a This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. null mouse has a behavioral obesity syndrome characterized combination of diets. The most known approaches are high by hyperphagia, hyperglycemia, hyperinsulinemia, reduced carbohydrate, high sucrose, high fructose, and HFDs. The mode metabolism, increased lean mass, and altered hepatic lipid of action for each diet, with emphasis on the affected biochemi- metabolism.40,41 It has been also shown that MC4-R/ mice cal pathways, has been beautifully reviewed by Wong et al52 and have elevated total plasma cholesterol and TG but tend to be is beyond the scope of this review. Here we will present some hypotensive rather than hypertensive.34,42 Cho et al from key characteristics of the main experimental diets. As far as HFD Yale University43 created a polygenic obesity-induced dia- is concerned, numerous modifications exist, with fat concen- betic mouse model (RCS10) by combining different obesity trations ranging from 20 to 60%, originating either from plants risk-conferring quantitative trait loci (eQTLs) from two dif- (e.g., corn, safflower, or olive oil) or animals (e.g., beef, tallow, ferent strains; NZO/HlLt and NON/Lt. This is a type 2 diabetes and lard).53 The common main outcome of HFD is the increased model accompanied by adult-onset obesity, hyperglycemia, formation of TG following re-esterification of free fatty IR, and increased hepatic, intramuscular and heart TG at acids after lipolysis.52 Interestingly, HFD-related outcomes are 13 weeks of age; however, data of the effect on blood fat-dose-dependent.54 Fructose is one of the monosaccharides pressure are lacking.43 alongside glucose and galactose and it is only an intermediary

hobssadHeotssVl 2 o /01©22.Tim.Alrgt reserved. rights All Thieme. 2020. © 6/2021 No. 121 Vol. Haemostasis and Thrombosis Table 1 Genetic CMS mouse models and their metabolic phenotype Update An Syndrome: Cardiometabolic

Mouse model Genetic modification Metabolic changes Source Obesity Hyperglycemia Insulin Hypertension Dyslipidemia resistance – ob/ob Autosomal recessive UU(strain dependent) U ✖ " Total cholesterol 30 32 mutation in leptin gene (LDL/HDL1) db/db Autosomal recessive mutation in UU(age, strain U ✖ " Total cholesterol 33 leptin receptor gene dependent) (LDL/HDL1) MC4-R / Targeted disruption U UU↓blood pressure " Hepatic TG, 40,41 of MC4-R gene (exacerbated " serum FFA on HFD) Ay/a Spontaneous U ↓Glucose tolerance U U " HDL 36,37 mutations in agouti gene (delayed onset) (HFD þ HS-induced) (delayed onset) / 22,24 Ldlr Deletion of Ldlr gene U ↓Glucose tolerance U ✖ " LDL al. et Aravani (HFD-induced) (HFD-induced) (HFD-induced) Apoe / Deletion of Apoe gene Generally ✖ Generally ✖ " Total cholesterol, 23 resistant resistant triglycerides, VLDL, LDL, L NZBWF1 Cross between New Zealand U ✖ UU – 47 black (NZB) mouse with the New Zealand white (NZW) mouse Adipo / 2–4weeks Adiponectin knockout UU U– " VLDL 45 on diet A-ZIP/F-1 Overexpression of A-ZIP/F ✖ UUUU48,50 in adipose tissue aP2-SREBP-1c Overexpression of SREBP-1c ✖ UU– " Hepatic TG 49 in adipose tissue RCS10 Combination of obesity risk loci on U UU– "Hepatic and 43 2 strains; NZO/HlLt and NON/Lt (adult onset) intramuscular TG POUND mouse Deletion of all leptin isoforms UU U– " Total cholesterol Charles River

Abbreviations: CMS, cardiometabolic syndrome; FFA, free fatty acids; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein. Note: U and ✖ indicate the presence or absence respectively, whereas “–“ indicates no data.

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Table 2 Diet-induced CMS mouse models, the diet treatment, and their metabolic phenotype

Mouse strain Diet treatment Metabolic changes Obesity Hyperglycemia Hypertension Dyslipidemia Male C57BL/6 J mice High-fat diet, 8 wk UU – U Male AKR/J (AKR) mice High-fat diet, 8 wk UU – U Male and female High-fat diet (60%), 20 wk UU – U C57Bl/6J mice Male and female A/J mice High-fat diet (60%), 20 wk U ✖ – U Male C57BL/6 mice High-fat diet, 16 wk UU – U Male C57BL/6 mice High-fat diet, 40 wk –– – U Female C57Bl/6 J mice High-fat diet, 24 wk U ––U C57BL/6 mice (wild type) High-fat diet, 13 wk – ✖ – U Ldlr / mice High-fat diet, 13 wk – U – U Female C57BL/6NTac mice High-fat diet, 12 wk UU – U Male C57BL/6 mice High-fat diet, 12 wk U ––U Male C57BL/6 J mice High-fat diet, 4 wk U ––U Male C57BL/6 mice High-fat diet, 16 wk UU – U Male NMRI mice Fructose drinking U ––– water (15%), 10 wk ✖ ––– Sucrose soft drink (10%), 10 wk ✖ ––– Noncaloric soft drink, 10 wk

Abbreviation: CMS, cardiometabolic syndrome. Note: U and ✖ indicate the presence or absence respectively, whereas “–“ indicates no data. Source: Adapted and reprinted with permission from Wong SK, Chin K-Y, Suhaimi FH, Fairus A, Ima-Nirwana S. Animal models of metabolic syndrome: a review. Nutr Metab. (Lond) 2016;13:65. molecule during glucose metabolism. Chronic fructose con- metabolic syndrome has also been reported. Glucocorticoids sumption is an established way to induce increased energy are hormones that regulate glucose homeostasis in various intake, body weight, adiposity, hypertriglyceridemia, hyperlip- ways such as gluconeogenesis in liver, whereas in skeletal idemia, hypertension, glucose intolerance, and decreased insu- muscle and WAT they decrease glucose uptake and utilization lin sensitivity in laboratory animals and can be more effective by antagonizing insulin response. Therefore, mice treated with than glucose or starch-feeding.52,55 According to Wu et al, a low glucocorticoids display glucose intolerance and reduced insu- dose of fructose in drinking water (10%) is sufﬁcient to induce lin sensitivity. Apart from body weight gain, they present CMSin animals.24 As far as sucrose (tablesugar)-enriched diet is dyslipidemia, central, liver and skeletal muscle fat accumula- concerned, it is widely used for induction ofobesity, wholebody tion, and hypertension.59 IR, hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. Amounts of sucrose supplementation range from 20 to 52,56 Newer CMS Mouse Models Used in 77%. However, sucrose appears to be less effective than the Cardiometabolic Research

equivalent amount of fructose in inducing CMS as it is a This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. disaccharide consisting of 50% fructose and 50% glucose. Dietary Interventions Besides, mouse response to daily sucrose consumption is Diet-induced obesity models are repeatedly used to assess the strain-dependent, affected in particular by polymorphisms in effect ofvarious factors on cardiometabolic phenotype. It seems the Tas1r3 sweet taste receptor gene.57 that HFD administered for various intervals is one of the most The effects of each diet or diet combination on mice are preferable interventions used to induce CMS. This approach summarized in ►Table 2. most frequently leads to weight gain, dyslipidemia, and IR. C57/ BL/6J mice fed with 60% HFD for 11 weeks were used as a model Drug/Chemically Induced CMS Mouse Models of obesity to study the effects of angiotensin (1–7). These obese Streptozotocin administration is widely used to induce diabe- mice exhibited high blood pressure and reduced insulin sensi- tes in mice. The mode of action for streptozotocin is via tivity.60 Similarly, Miranda et al utilized diet-induced obesity to destruction of the β pancreatic cells. Streptozotocin is a glucose investigate the effect of cannabinoid receptor 1 antagonists. In analogue that accumulates in β pancreatic cells via the GLUT2 this study, mice were fed with 60% HFD for a longer period glucose transporter. Type 1 diabetes manifestation occurs in (16 weeks). HFD increased body weight, fasting glucose, and IR, mice treated with Streptozotocin.58 Glucocorticoid-induced but also promoted adipose tissue inﬂammation characterized

by inflammatory M1 macrophage infiltration.61 Another study which can sway the balance between protection and lipotox- confirmed the HFD-induced M1–M2 imbalance in WAT and the icity,80,81 sex-specific responsiveness to HFD82 and strain spec- abnormalities in glycemic indexes and lipid profiles.62 ificity to sugar consumption,57 behavioral effects83 and Guo et al63 fed 2-month-old male C57BL/6 mice with either intramyocellular lipid accumulation causing muscle defects.84 HFD (45% energy from fat) including 7% sucrose or low-fat diet Although obesity-related complications such as IR, nonal- (LFD; 10% energy from fat). Control mice fed LFD were com- coholic fatty liver disease, and CVD share common pathophys- pared withvarious HFD groups which were supplemented with iology mechanisms such as increased tissular lipid deposition, one of three fatty acids (EPA, DPA, and DHA) or none, after hypoxia, and low-grade inflammation, different lipid species 6 weeks of intervention. The HFD group with no addition of and groups are implicated in the aforementioned processes.85 fatty acids showed gathered characteristics of the CMS, in that For instance, in the case of atherosclerosis and CVD, it is widely mice were significantly obese with increased liver weight and accepted that the presence of ApoB-containing lipoproteins epididymal adiposity, fatty liver, increased hepatic total choles- such as very low-density lipoprotein (VLDL), LDL, and lipopro- terol, high serum glucose, alanine transaminase, reduced adi- tein-a is of determinant importance for the pathogenesis of ponectin, and IR. Additionally, upregulation of inflammatory such complications.86 The adipose tissue, on the other hand, is genes in the TLR-4/NF-κB pathway was observed. Supplemen- predominantly characterized by the presence of triacylglycer- tation of long-chain omega-3 polyunsaturated fatty acids could ols rather than cholesterol or phospholipids and, during reverse the above effects. Blood pressure was not assessed in obesity, specific triacylglycerol signatures, defined by different this model.63 Leptin levels were also elevated after 8 weeks of fatty acid compositions, are associated to the development of HFD in a different study.64 Similar phenotypes were reported by IR.87 Cholesterol and especially cholesterol esters are consid- – several studies using HFD as a mean of mimicking the CMS.65 69 ered important inducers of nonalcoholic fatty liver disease, CMS-related serum biomarkers were investigated in HFD (60%) independently of obesity and metabolic dysregulation.88 Nev- fed mice for an extended period (36 weeks). Besides increased ertheless, the progression of the disease into nonalcoholic body weight, dysregulated lipid metabolism (high total choles- steatohepatitis in both humans and mice depends on the terol and TG), raised blood glucose, insulin, glycated hemoglo- existence of specific long-chain fatty acids that are toxic to bin (HbA1c), and brain natriuretic peptide, there were also the hepatocytes.89 Considering that the different diets that are abnormalities in renin–angiotensin system. This was suggested used in animal models to provoke CMS trigger the emergence by high serum angiotensin-converting enzyme, angiotensin II of different metabolic signatures in vivo and finally simulate (Ang-II), Ang-II type 1 receptor, and aldosterone. Cardiac different CMS-related complications, and not all of them remodeling was also observed in the HFD group compared together, exceptional caution should be taken when animal with normal diet (10% fat).70 A combination of diet and drug- models of CMS are used for the study of CVD. induced CMS has also been tested. Here mice were maintained on 60% HFD for 5 to 15 weeks alongside nitric oxide synthase Genetic Manipulations inhibitor L-NAME. This double hit model71,72 developed several In addition to diet-induced strategies, genetic mouse models aspects of CMS, as evident by obesity, hyperglycemia, reduced have been extensively used as illustrated in the first part of insulin sensitivity, impaired glucose clearance (heightened this review. Genetic manipulations include either manipu- fasting glucose, HbA1c), hypertension, and endothelial dysfunc- lation of known metabolic pathways or exploration of new tion. Various diet modifications have been also described, such genes that have been shown to be associated with human as high fat (60%) diet with salt addition (7.25% NaCl), which was CMS. This approach allows researchers to dissect the genetic sufficient to cause most of the aspects of CMS including component of CMS, induce more severe phenotypes, and in diabetes, hypertension, and secondary features such as cardiac some occasions shorten the study duration compared with dysfunction and vascular remodeling.73 A combination of high long-term feeding protocols. Such an example is the human fat (35%)–high sucrose (34%) long-term (4 months) feeding cholesteryl ester transfer protein (CETP), which has no protocol caused body weight gain, impaired glucose homeosta- homologous gene in mouse and has been found to be 90

sis, increased hepatic and myocardial TG content, as well as associated with human CMS. CEPT is responsible for the This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. cardiac dysfunction in male C57BL6R mice.74 All the above exchange of TG and cholesterol esters between LDL and VLDL studies indicate that HFD is a reliable and simple protocol that particles and HDL. CETP activity is associated with reduced induces reproducible phenotypes. However, something that HDL and increased risk of atherosclerosis. There is no mouse should be taken into consideration when it comes to HFD homologue of this gene, therefore overexpression of the protocols is that mice may respond differently according to human CETP has been applied to mimic the human condi- their genetic background75,76 or their gut microbiota adapta- tions. In several studies, CETP overexpression is combined tion to HFD. This adaptation involves transcriptional changes in with Apoe deﬁciency conferring reduced HDL and reduced hepatic miRNAs leading to a divergent metabolic phenotype. clearance of TG-rich lipoproteins. APOE3-Leiden.CETP Subsequently, one can claim that hepatic lipid metabolism is mouse is a model combining obesity, IR, and hyperlipidemia modulated according to the response to HFD.77,78 Other when treated with HFD and fructose. It is responsive limitations regarding HFD include disparities in phenotypes to antidiabetic and lipid-lowering drugs evidenced by sometimes attributed to the modulation of the HFD protocol (i. improved glucose regulation, IR, and dyslipidemia.91 More- e., duration of intervention, age of mice)79 and the diet compo- over, transcriptome analysis revealed that these mice when sitions particularly the type of fat and amount of carbohydrates, fed HFD comprise distinct hepatic gene signatures and serum

Thrombosis and Haemostasis Vol. 121 No. 6/2021 © 2020. Thieme. All rights reserved. Cardiometabolic Syndrome: An Update Aravani et al. 709 miRNAs.92 Hemizygous CETP expression combined with Ldlr induced obesity (60% HFD) was sufficient to cause arterial haploinsufficiency was also used as a model that reflects the hypertension, but not obesity (compared with wild types on lipoprotein metabolism and the risk of cardiometabolic HFD), increased area under the curve during glucose tolerance disease in humans. These transgenic mice have perturbed test which was maintained from 8 to 18 weeks of feeding, and lipid profiles showing increased LDL and decreased HDL. raised blood insulin.105 Glucose was not altered as a function of CETP expression and Another genetic model utilizing the neuropeptide Y (NPY) blood pressure and body weight data were not available. was first described in 2008.106 NPY is a co-transmitter Thus this model lacks some of the main components of expressed in the brain and released alongside noradrenaline CMS.93 in response to sympathetic stimulation. It is also expressed in The Ldlr-deficient mouse treated with HFD is still commonly neurons innervating the vasculature cardiomyocytes and used and seems to be quite consistent as far as the lipid endocardium, and is involved in physiological processes phenotype, hepatic inflammation, and atherosclerosis devel- including vasoconstriction, cardiac remodeling, and angio- – opment are concerned.94 97 Ldlr / mice fed high fat sucrose genesis. Overexpression of NPY in noradrenergic neurons of enriched diet with 0.15% cholesterol for 12 weeks developed the central and peripheral nervous system leads to increased diet-induced obesity, increased fasting insulin and glucose WAT weight (subcutaneous, epididymal, and retroperitoneal levels, and dyslipidemia characterized by increased TG and fat pads) but not overall body weight, increased liver TG, and total cholesterol. Accelerated liver inflammation was also fasting glucose. These changes occurred in the absence of detected by elevated expression of inflammatory genes such modified diet. The relevance of NPY in cardiometabolic as Emr1, Tnfa, ll-6, Ccl2 as well as susceptibility to atherosclero- animal modeling is also supported by human studies, where sis. However, blood pressure was not evaluated in this mouse genetic polymorphisms found in NPY receptors (Y1, Y2, Y5) model.94 Apoe-deficient mice appeared less frequently in the are associated with cardiometabolic risk factors and early- literature search we conducted. Crossing of heterozygous aro- onset CVD risk.107,108 Deletion of protein kinase D2 (PRKD2) matase-deficient mice (Arþ/)ontheApoe/ background was triggers hyperinsulinemia which precedes IR and precipi- used to generate metabolically altered mice (MetS-Tg). The tates metabolic changes. Increased β-cell insulin secretion double knockout animals displayed central obesity, increased was mediated by increased expression and activation of L- body weight, elevated serum, and increased blood pressure. Ca2þ channels and subsequent high glucose and membrane Glucose and insulin tolerance tests revealedglucose intolerance depolarization. The authors have focused on insulin secre- and IR. Detection of cytokines such as TNF-a, IL-6, and CRP tion regulation rather than lipid metabolism and blood indicates an inflammation state.98 The same group reported pressure data which are not reported.109 almost identical results for MetS-Tg mice in a different study, Besides the known obesity-induced diabetes models, ob/ob which were characterized by increased body weight compared and db/db, which are widely used with or without a diet – with wild types, no differences in plasma glucose but in glucose combination,110 113 a very recent study has designed a new area under the curve, and higher mean arterial blood pres- mouse model, which recapitulates maturity-onset diabetes of sure.99 The induction of hyperlipidemia has been traditionally the young (MODY).114 MODY4 mouse is essentially a pancre- achieved by time-consuming generation of knockout trans- atic and duodenal homeobox 1 (Pdx1) haploinsufficient ani- genics of Ldlr and Apoe. Recently, hyperlipidemia has been mal. Pdx1 is a transcription factor that regulates β-pancreatic attempted via adeno-associated-virus-8 (AAV8)-mediated cell maturation. Pdx1þ/ mice were further challenged by overexpression of PCSK9.100 A single intravenous femoral injec- genetic inhibition of the stress-responsive IKK/NF-κBsignaling tion of viral particles containing the human PCSK9(DY) gene, pathway resulting in a double transgenic IKK2-DNPdx1 ani- which is a gain-of-function mutation of PCSK9 under the liver- mal. Transgenics presented rapid onset of diabetes character- specific promoter HCR-hAAH, led to stable hepatic expression ized by hyperglycemia, hyperinsulinemia, and loss of β-cell of this mutant protein. Mice expressing the mutant PCSK9 had mass.114 Although this is a model of diabetes, it could be increased serum cholesterol and particularly LDL from day 30 potentially combined with high fat or other dietary interven- 100 up to 1-year postinjection, under normal feeding conditions tion to introduce hyperlipidemia and/or obesity. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. and much earlier (day 7) when treated with HFD as reported by others.101 Consistent with the elevated fraction of LDL, the Other Models for CMS Simulation hepatic expression of Ldlr was significantly reduced. HFD The model of maternal separation and early weaning (MSEW) exacerbated the observed hyperlipidemia and atherogenicity. together with high fat feeding predispose female offspring to an The potency of the adenovirus-mediated PCSK9 transexpres- early onset of cardiometabolic risk factors, including hyper- sion was also confirmed in three different genetic mouse insulinemia, glucose intolerance, and hypercholesterolemia as strains. This fast and efficient way of introducing hypercholes- well as increased weight and fat mass compared with MSEW terolemia in mice without the need ofgenetic manipulation has and non-MSEW male counterparts. Both male and female – since been employed by several groups.102 104 MSEW mice fed HFD also exhibited an increase in blood Genetic ablation of GC-Aguanylyl cyclase-A (GC-A) receptor pressure compared with controls, non-MSEW.115 Other studies in β pancreaticcells is a novelgenetic mouse model used for the that link maternal feeding status and the cardiometabolic investigation of the role of atrial and brain-type natriuretic profile of offspring have been reported. Saad et al allocated peptides and its GC-A receptor in glucose homeostasis. The pregnant C57BL/6Jmiceto fructosesolutionor water from day 1 disruption of GC-A receptor in β-cells combined with diet- of pregnancy. The fructose protocol promoted hypoglycemia as

indicated by intraperitoneal glucose testing in both sexes and The above studies suggest that the prenatal and perinatal higher mean arterial pressure compared with controls but had periods are as important as the postnatal lifestyle for the no effect on lipid levels. Moreover, there was a female-specific onset of CMS and—as highlighted by others—it is important effect on various other parameters following fructose intake to understand the long-term effects of treatment during compared with the control group. Female mice had higher pregnancy as it can have significant effects on offspring.120 weights with visceral adiposity, hepatic fat accumulation, IR, It is also worth mentioning that wild-derived inbred and high serum leptin.116 Similarly, another group concluded strains have been suggested to be informative about our that maternal diet-induced obesity coupled with postweaning knowledge of CMS. These inbred strains arose from inbreed- obesogenic diet worsened offspring hyperinsulinemia, hyper- ing of wild mice caught around the world. Karunakaran and leptinemia, fat deposition, hypertension risk, and cardiac fibro- Clee have extensively reviewed the characteristic of these sis.117 A recent study has implicated maternal diet in offspring wild-derived species (Cast, Molf, PWD, PWK, Spret, WSB), malfunctioning metabolism. Maternal high-fructose feeding which are generally obesity-resistant but they show a spec- caused multigenerational hypertension, which was character- trum of abnormalities in lipid and glucose metabolism.21 ized by increased mRNA expression of the RAAS genes, as well Finally, in ►Table 3 we present additional mouse models as the expression of renin in the kidneys. The authors conclude that carry the main CMS components after searching the MGI that high fructose intake during pregnancy activated the RAAS database for mouse models under the phenotype metabolic – and epigenetically induced changes in the cardiometabolic syndrome.121 134 Moreover, the MGI search confirmed already system of the offspring.118 While maternal nutrition status mentioned models such as HFD-induced, ob/ob,KKAy/a, strep- can affect the progeny, a different study reported that paternal tozotocin-induced, genetic strains (PWD, PCD1), NPY, and cholestasis (10 weeks of 0.5% cholic acid-supplemented diet triple knockouts (ob/ob; Apoe/ on ApoB100 background). prior to mating) resulted in metabolic defects in male offspring. Male pups of cholestatic fathers further treated with western Conclusion diet for 18 weeks had raised fasting insulin, hepatic TG content, and serum cholesterol levels compared with diet-matched CMS is a global epidemic with increasing prevalence. Due to controls. At 25 weeks of age, male offspring showed hyperten- its complex etiology—a combination of genetic and environ- sion as well.119 mental factors—and pathophysiology, it is challenging to

Table 3 Additional genetic CMS mouse models as reported in MGI database and their metabolic phenotype

Mouse model Genetic modification Metabolic changes Source GNB3/þ BAC transgenic mice that carry " Fasting plasma glucose, insulin, and C-peptide, 121 an extra copy of human GNB3 triglycerides, cholesterol, phospholipids, and glucose intolerance SNAP-25b Targeted deletion of SNAP-25b Hyperglycemia, liver steatosis, and adipocyte hyper- 122 trophy (HFD exacerbated) Het-MλKO Heterozygousmuscle-speciﬁc Hyperglycemia, hyperinsulinemia, abdominal obesity, 123,124 PKC-λ knockout hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia Tyk2 / Tyrosine kinase 2 knockout Obesity, " plasma insulin, cholesterol, and FFA 125 AdiposeENPP1-TG Targeted overexpression of " Plasma FFA, triglycerides, glucose, insulin, fatty liver 133 human ENPP1 in adipocytes (HFD-induced) Ankrd26 Partial inactivation of the Obesity, " plasma insulin 126 Ankrd26 gene This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. PrRP-deﬁcient mice Targeted disruption of PrRP gene Late-onset obesity and adiposity, " blood glucose 127 Timo/Timo Targeted disruption of Bdnf gene Obesity, hepatic steatosis, " leptin, insulin, glucose 128 cholesterol, LDL cholesterol Neil1/ Targeted deletion of Neil1 Severe obesity, dyslipidemia, fatty liver disease, and 129 tendency to hyperinsulinemia GPCR12 KO Deletion of GPCR12 gene " Body weight, body fat mass, hepatic steatosis, 130 dyslipidemia RS1 / Targeted deletion of RS1 gene Obesity, " total fat, serum cholesterol, and leptin 131 PLSCR3 / Targeted deletion of PLSCR3 gene " Abdominal fat, FFA, triglycerides, and leptin, ↓ leptin, 132 insulin resistance, glucose intolerance 11β HSD-1 TG Selective overexpression of Visceral obesity (HFD exacerbated) insulin resistance, 134 11β HSD-1 in adipose hyperlipidemia, hyperphagia despite hyperleptinemia

Abbreviations: CMS, cardiometabolic syndrome; FFA, free fatty acids; HFD, high-fat diet; LDL, low-density lipoprotein; MGI, mouse genome informatics.

Thrombosis and Haemostasis Vol. 121 No. 6/2021 © 2020. Thieme. All rights reserved. Cardiometabolic Syndrome: An Update Aravani et al. 711 address effective therapies. Animal models have traditionally most importantly, the differences between human and ani- guided research and shed light on the involved mechanisms mal models.142 and candidate therapeutics. In the first part of the review, we summarized the best Funding characterized mouse models for CMS; the last comprehen- This work was funded by a grant from the European sive reviews focusing on CMS mouse models date back in Foundation for the Study of Diabetes (EFSD) to A.C. 2010.34,135 In the second part we reviewed the PubMed and MGI database and described mouse models that have been Conflict of Interest developed in the last 10 years. Also, we identified the None declared. models that are repeatedly used, confirming their utility and contribution to the field. We conclude that ob/ob and db/db mice are the most common obesity and IR models, References whereas Ldlr / and Apoe / are widely used to induce 1 Kylin E. Studien ueber das hypertonie-hyperglyka. Zentralblatt – hyperlipidemia. These mice have been extensively used Fuer Inn Med 1923;44:105 127 2 Vague J. The degree of masculine differentiation of obesities: a either as a single transgenic or combined with a different factor determining predisposition to diabetes, atherosclerosis, background with or without diet treatment. As far as diet- gout, and uric calculous disease. Am J Clin Nutr 1956;4(01): induced models are concerned, we found that HFD (40–60%) 20–34 with modifications (sucrose, salt addition) is the preferred 3 Alberti KGMM, Eckel RH, Grundy SM, et al; International protocol, which generally leads to increased body weight, Diabetes Federation Task Force on Epidemiology and Preven- hyperlipidemia, and IR. tion Hational Heart, Lung, and Blood Institute American Heart Association; World Heart Federation International Atheroscle- The significance of mouse as a proxy organism to study rosis Society International Association for the Study of Obesity. the human CMS has been extensively documented through Harmonizing the metabolic syndrome: a joint interim state- numerous studies. The generation of genetically engineered ment of the International Diabetes Federation Task Force on mouse models and the idea of altered diet consumption Epidemiology and Prevention; National Heart, Lung, and Blood date back to early 1990s. An important advantage is the Institute; American Heart Association; World Heart Federation; availability of inbred strains, which provide a constant International Atherosclerosis Society; and International Asso- ciation for the Study of Obesity. Circulation 2009;120(16): genetic background (within strains) that allows us to study 1640–1645 – – the effects of various manipulations (gene gene, gene 4 Grundy SM, Brewer HB Jr, Cleeman JI, Smith SC Jr, Lenfant environmental) on CMS development. However, there are CAmerican Heart Association National Heart, Lung, and Blood defined genetic differences among different inbred strains, Institute. Definition of metabolic syndrome: report of the which can alter their responsiveness threshold. In the National Heart, Lung, and Blood Institute/American Heart Asso- ciation conference on scientific issues related to definition. context of CMS, it is proven that susceptibility to athero- Circulation 2004;109(03):433–438 sclerosis, hyperlipidemia, hypertension, and diet adaptation 5 Castro JP, El-Atat FA, McFarlane SI, Aneja A, Sowers JR. Cardio- 31,136–138 varies among different mouse strains. Thus, this metabolic syndrome: pathophysiology and treatment. Curr should be taken into account when interpreting or compar- Hypertens Rep 2003;5(05):393–401 ing inter-study results. Furthermore, for diet-induced CMS 6 Grundy SM, Cleeman JI, Daniels SR, et al; American Heart the selection of proper control diet is crucial as cardiome- Association National Heart, Lung, and Blood Institute. Diagnosis and management of the metabolic syndrome: an American tabolic alterations were found to be underestimated in low 139 Heart Association/National Heart, Lung, and Blood Institute fat diet-treated controls. Another emerging issue that Scientific statement. Circulation 2005;112(17):2735–2752 needs to be addressed is the impact of sex in the study of 7 Mottillo S, Filion KB, Genest J, et al. The metabolic syndrome and CMS. Traditionally, male mice are preferred over females cardiovascular risk a systematic review and meta-analysis. J Am because of the known development of a more severe disease Coll Cardiol 2010;56(14):1113–1132 phenotype and because of the concern that the estrous 8 Yates KF, Sweat V, Yau PL, Turchiano MM, Convit A. Impact of metabolic syndrome on cognition and brain: a selected review of

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